Dual-functional nonfouling surfaces comprising target binding partner covalently coupled to polymer attached to substrate

ABSTRACT

Dual-functional nonfouling surfaces and materials, methods for making dual-functional nonfouling surfaces and materials, and devices that include dual-functional nonfouling surfaces and materials. The dual-functional surfaces are nonfouling surfaces that resist non-specific protein adsorption and cell adhesion. The dual-functional surfaces and materials include covalently coupled biomolecules (e.g., target binding partners) that impart specific biological activity thereto. The surfaces and materials are useful in medical diagnostics, biomaterials and bioprocessing, tissue engineering, and drug delivery.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/396,128, filed Dec. 30, 2016, which is a division of U.S. patentapplication Ser. No. 14/470,775, filed Aug. 27, 2014, which is adivision of U.S. patent application Ser. No. 12/493,649, filed Jun. 29,2009, which is a continuation of International Patent Application No.PCT/US2007/089236, filed Dec. 31, 2007, which claims the benefit of U.S.Provisional Application No. 60/882,821, filed Dec. 29, 2006. Eachapplication is expressly incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Contract No.N000140410409 awarded by the Office of Naval Research. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Surfaces that are resistance to protein adsorption and cell adhesion arecritical for the development of biosensors with high specificity andbiomaterials with superior compatibility. Currently, oligo(ethyleneglycol) (OEG)- or polyethylene glycol (PEG)-based materials such as OEGself-assembling monolayers (SAMs) or poly(OEG methacrylate) are the mostcommonly used nonfouling materials. Recent studies attribute thenonfouling properties of OEG SAMs to their strong hydration capabilitiesand well-packed structures. Another commonly used nonfouling material isbiomimetic phosphoryl choline (PC)-based materials containing PCheadgroups, which are found in the outside layer of cell membranes.Polymers or surfaces modified with PC have been shown to reduce proteinadsorption. Recently, applicants have shown that poly(sulfobetainemethacrylate) (polySBMA)-grafted surfaces have low protein adsorptionwhen they are grafted from a surface via the surface-initiated atomtransfer radical polymerization (ATRP) method.

Many applications of surface plasmon resonance (SPR) analysis requireimmobilization of antibodies on the SPR sensor. SPR sensors havingimmobilized antibodies have been used to detect analytes from complexbiological solutions. The elimination of non-specific protein adsorptionwhile maximizing the analyte signal is critical for SPR sensors.Different polymers, most of them with carboxylic acid groups, such ascarboxymethylated dextran, carboxymethylated hyaluronic acid,polyacrylic acid and DL-polylactic acid, have been immobilized on theSPR gold surface. However, all these polymers are not protein resistantmaterials, which means they could adsorb non-specific proteins fromcomplex biological solutions and mask the signal from analytes ofinterest. Usually, proteins such as BSA have been used to block asurface after the antibody immobilization, which can decreasenon-specific binding, but can lead to low efficiency and some unwantedreactions for in vivo testing.

For biosensor or biomaterial applications, it is desirable to have anonfouling surface or material as a background while presenting anabundance of functional groups for ligand immobilization. Mixedcarboxylic (or amino) and hydroxyl-terminated oligo(ethylene glycol)(OEG) self-assembled monolayers (SAMs) or carboxymethyl dextran polymersare currently used in biosensor applications. Copolymers containingdecoratable segments and nonfouling polyethylene glycol (PEG) polymersor low-fouling poly(hydroxyethyl methacrylate) orpoly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) segments havebeen prepared to control cell and tissue responses. However, extra (orunreacted) functional groups can compromise the specificity of a sensoror the biocompatibility of a biomaterial.

Therefore, there is a need for a nonfouling biomaterial with activefunctional groups for protein or ligand immobilization. The presentinvention seeks to fulfill these needs and provides further relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides dual-functional nonfouling surfaces,methods for making dual-functional nonfouling surfaces, and devices thatinclude dual-functional nonfouling surfaces.

In one aspect, the invention provides substrates with nonfoulingsurfaces having biomolecules covalently coupled thereto. In oneembodiment, the substrate has a modified surface that includes

(a) a plurality of polymers attached to a surface of a substrate, whereeach polymer includes a plurality of carboxylic acid groups and aplurality of positive charged groups, and where each polymer issubstantially electronically neutral; and

(b) a plurality of target binding partners, where the target bindingpartners are covalently coupled to a plurality of the polymers, andwhere the target binding partner has affinity toward a target molecule.

In one embodiment, the plurality of polymers are covalently coupled tothe surface. In one embodiment, the plurality of polymers are covalentlycoupled to the surface through a monolayer comprising a plurality ofalkylene moieties and form brushes on the surface. In one embodiment,the polymers are poly(carboxybetaines).

In another aspect, the invention provides methods for modifying surfacesto provide nonfouling surfaces having biomolecules covalently coupledthereto. In one embodiment, the method includes covalently coupling aplurality of target binding partners to a plurality of polymers attachedto a surface, where the target binding partner has affinity to a targetmolecule, and where each polymer comprises a plurality of carboxylicacid groups and a plurality of positive charged groups, and where eachpolymer is substantially electronically neutral.

In one embodiment, covalently coupling the plurality of target bindingpartners to the plurality of polymers includes forming an amide linkagebetween the polymers and the target binding partners. In one embodiment,covalently coupling the plurality of target binding partners to theplurality of polymers comprises converting a portion of carboxylic acidgroups to activated esters and reacting the activated esters with targetbinding partners having amino groups. In one embodiment, the activatedesters are N-hydroxysuccinimide esters. In one embodiment, the polymersare poly(carboxybetaines).

In another aspect, materials are provided that are nonfouling and thathave biomolecules covalently coupled thereto. In one embodiment,crosslinked polymers are provided. In another embodiment, blockcopolymers are provided.

The crosslinked polymers have a plurality of target binding partnerscovalently coupled thereto. The crosslinked polymers comprises aplurality of carboxylic acid groups and a plurality of positive chargedgroups, where the polymers are substantially electronically neutral, andwhere the target binding partner has affinity toward a target molecule.In one embodiment, the crosslinked polymer is a crosslinkedpoly(carboxybetaine). In one embodiment, the crosslinked polymer is ahydrogel. Methods for making the crosslinked polymers are also provided.

The block copolymers include a first hydrophilic block and a secondhydrophobic block. The block copolymer has a plurality of target bindingpartners having affinity toward a target molecule covalently coupledthereto. The hydrophilic block comprises a plurality of carboxylic acidgroups and a plurality of positive charged groups, and the hydrophilicblock is substantially electronically neutral. In one embodiment, thehydrophilic block is a poly(carboxybetaine). In one embodiment, theblock copolymer is in the form of a microparticle or a nanoparticle.Methods for making the block copolymers are also provided.

In other aspects, devices that include dual-functional nonfoulingsurfaces are provided.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIGS. 1A and 1B are schematic illustrations of representativedual-functional surfaces of the invention;

FIG. 2 shows the synthesis of a representative zwitterionic polymer withpositively-charged backbone;

FIG. 3 is a schematic illustration of a representative method forpreparing a surface coated with a poly(carboxybetaine methylacrylate)(CBMA) by surface initiated atom transfer radical polymerization (ATRP)and having a protein covalently coupled to the polymer;

FIG. 4 shows the adsorption of 1 mg/mL fibrinogen, 1 mg/mL lysozyme, and20 ug/mL anti-hCG from PBS solutions (150 mM and pH 7.4) onpoly(CBMA)-grafted surfaces by SPR measurements;

FIG. 5 shows the adsorption of 1 mg/mL fibrinogen, 1 mg/mL lysozyme, and20 ug/mL anti-hCG from PBS solutions (150 mM and pH 7.4) onpoly(CBMA)-grafted surfaces with immobilized anti-hCG by SPRmeasurements;

FIG. 6 shows the adsorption of 1 mg/mL fibrinogen, 1 mg/mL lysozyme, and40 ug/mL anti-hCG from PBS solutions (150 mM and pH 7.4) onpoly(CBMA)-grafted surfaces with immobilized anti-hCG by SPRmeasurements;

FIG. 7 shows the effect of antibody immobilization buffer on finalantibody immobilization level on an activated poly(CBMA) coated surface;

FIG. 8 shows a typical response to immobilization of antibody at pH 8.0on a representative poly(CBMA) coated surface;

FIG. 9 compares the detection of activated cell leukocyte adhesionmolecule (ALCAM) on a surface immobilized with antibodies againstactivated cell leukocyte adhesion molecule (anti-ALCAM) and a referencesurface with immobilized antibodies against Salmonella (anti-Salm) in50% plasma;

FIG. 10 is a sensorgram corresponding to the immobilization ofanti-ALCAM and anti-Salm onto TSTU-activated poly(CBAA)-grafted surfacewith the running buffer having a antibody concentration of 10 ug/mL and50 ug/mL, consecutively, in 10 mM sodium acetate at pH 5.0;

FIG. 11A shows the control of nonfouling properties of poly(CBAA) afterdeactivation of residual carboxylic acid groups by injection offibrinogen in PBS with concentration of 1 mg/mL; and FIG. 11B comparesthe sensor response to ALCAM binding (100 ng/mL) in measuring(anti-ALCAM) and reference (anti-Salm) channels;

FIGS. 12A and 12B are microscopic images of bovine aortic endothelialcell (BAEC) adhesion after 24 h, FIG. 12A shows BAEC adhesion oninactivated crosslinked poly(CBMA) hydrogel incubated in a fibronectinsolution for 24 hours; and FIG. 12B shows BAEC adhesion onEDC/NHS-activated poly(CBMA) hydrogel incubated in a fibronectinsolution for 24 hours;

FIG. 13A is a schematic illustration of a representative platform andmethod of the invention for protein assays, FIG. 13B is a schematicillustration of a conventional platform for protein assays;

FIG. 14 compares SPR sensorgrams for a representative CBAA polymersurface of the invention useful for protein assays showing superlowfouling of the surface to complex media including 100% serum, 100%plasma, and 1 mg/ml fibrinogen;

FIG. 15 is a spatially-resolved image of a representative proteinmicroarray of the invention from surface plasmon resonance (SPR)imaging, intensity changes from dark to bright represent protein bindingon the surface, anti-hCG and anti-Salmonella proteins were printed in500 μm spots using a microcontact printing robot, column 1,3,4, and 6are anti-hCG and column 2 and 5 are anti-Salmonella; and

FIG. 16 compares SPR sensorgrams for a representative CBAA polymersurface of the invention useful for specific detection of 10 mg/mL hCGon an anti-hCG protein spot, no detection is observed on the referenceanti-Salmonella protein spot or the background.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides dual-functional nonfouling surfaces,methods for making dual-functional nonfouling surfaces, and devices thatinclude dual-functional nonfouling surfaces. The dual-functionalsurfaces of the invention are surfaces that are nonfouling (e.g.,low-fouling or superlow-fouling) surfaces that resist non-specificprotein absorption and cell adhesion. The dual-functional surfaces ofthe invention also include covalently coupled biomolecules (e.g., targetbinding partners) that impart specific biological activity to thesurfaces. Because the dual-functional surfaces of the invention arenonfouling and include immobilized biomolecules, these surfaces that areuseful in any method or device that requires sensitive specific bindingor specific binding measurement and at the same time requires resistanceto non-specific protein binding and cell adhesion. The dual-functionalsurfaces of the invention are useful in medical diagnostic applications,biomaterials and tissue engineering, and drug delivery.

In one aspect of the invention, dual-functional surfaces are provided.The surfaces have the dual function of resisting or preventingnon-specific protein adsorption and cell adhesion and at the same timepermitting specific binding to a target molecule.

The surfaces of the invention are nonfouling, which means that thesurfaces resist or prevent non-specific protein adsorption and celladhesion. Non-specific protein adsorption can be measured by determiningthe level of fibrinogen adsorption (i.e., the amount of fibrinogen thatadsorbs to the surface per unit area). The surfaces of the inventionadsorb less than about 30 ng/cm² fibrinogen. In one embodiment, thesurfaces have a fibrinogen adsorption less than about 10 ng/cm²; in oneembodiment, the surfaces have a fibrinogen adsorption less than about 5ng/cm²; in one embodiment, the surfaces have a fibrinogen adsorptionless than about 3 ng/cm²; and in one embodiment, the surfaces have afibrinogen adsorption less than about 0.3 ng/cm².

The nonfouling nature of the surfaces of the invention are due to thematerials that make up the surfaces and their packing densities. In thepractice of the invention, nonfouling surfaces are prepared fromsurfaces by coating or covalently coupling a dual-functional material toa surface of a substrate. Thus, the surfaces of the invention aremodified surfaces (or coated surfaces).

The dual-functional materials useful for making the surfaces of theinvention are materials that impart nonfouling properties to thesurfaces and that also include a functional group or groups suitablyreactive to immobilize biomolecules (e.g., proteins such as antibodies).Representative functional groups for these dual-functional materialsinclude carboxylic acid groups and amino groups, among others. Asdescribed in detail below, in one embodiment, the dual-functionalmaterials are zwitterionic materials that can be attached (e.g.,covalently coupled) to a surface and that have terminal carboxylic acidgroups. These terminal carboxylic acid groups can be activated forcoupling to one or more amino groups present in a biomolecule. In thisembodiment, the carboxylic acid groups are converted to active esters(e.g., N-hydroxysuccinimide esters) and then reacted with one or moreamino groups (e.g., amino group of a lysine residue of a protein) toform an amide bond thereby immobilizing the protein to the surface toprovide a dual-functional surface of the invention.

The nonfouling function of the surface is imparted to the surface by thematerials coupled to the surface. As noted above, these materialsinclude zwitterionic materials. Suitable materials useful in making thesurfaces include the polymers and copolymers described inPCT/US2006/028988 (Superlow-Fouling Sulfobetaine and CarboxybetaineMaterials and related Methods), filed Jul. 25, 2006; PCT/US2007/075409(Mixed Charge Copolymers and Hydrogels), filed Aug. 7, 2007; and U.S.application Ser. No. 60/989,073 (Cationic Polycarboxybetaine Esters),filed Nov. 19, 2007, each expressly incorporated herein by reference inits entirety.

In one embodiment, the invention provides a substrate having a modifiedsurface that includes

(a) a plurality of polymers attached to a surface of a substrate,wherein each of the polymers comprises a plurality of carboxylic acidgroups and a plurality of positive charged groups, and wherein eachpolymer is substantially electronically neutral; and

(b) a plurality of target binding partners, wherein the target bindingpartners are covalently coupled to a plurality of the polymers, andwherein the target binding partner has affinity toward a targetmolecule.

In this embodiment, the materials making up the modified surfaceincludes polymers having a plurality of carboxylic acid groups and aplurality of positive charged groups. The polymers can be homopolymersin which the polymer includes repeating units that include bothcarboxylic acid groups and positively charged groups. The polymers canalso be copolymers that include two types of repeating units: repeatingunits that include carboxylic acid groups and repeating units thatinclude positively charged groups. The positively charged groups can bependant groups (i.e., pendant from the polymer backbone) or can be inthe polymer backbone. The polymers are substantially electronicallyneutral. As used herein, the term “substantially electronically neutral”refers to the polymer having substantially equal numbers of positive andnegative charges. It will be appreciated that carboxylic acid groups(—CO₂H) depicted and described herein are considered to be carboxylategroups (—CO₂ ⁻) for the purpose of determining a polymer's electronicneutrality. The electronic neutrality of the polymers making up themodified surfaces of the invention are responsible, in part, for thenonfouling properties of the surfaces of the invention.

In this embodiment, the polymers useful in the invention, and thesurfaces modified to include them, include carboxylic acid groups andcertain of these carboxylic acid groups are available for furtherchemical reaction, specifically the immobilization of biomolecules(e.g., target binding partners) to the polymers and, therefore, themodified surface. As noted above, in certain embodiments, biomoleculeshaving available amino groups (—NH₂) can be covalently coupled to thepolymers' available carboxylic acid groups to immobilize the biomoleculethrough stable amide linkages (—CONH—). In the practice of theinvention, a portion of the polymers having available carboxylic acidgroups are activated for coupling and then covalently coupled to thebiomolecules to provide surface-immobilized biomolecules. The extent ofbiomolecules immobilized on the surfaces of the invention can be readilycontrolled through the reaction conditions (e.g., extent of carboxylgroup activation and concentration and amount of biomolecule exposed tothe activated surface for immobilization. A schematic illustration of arepresentative nonfouling surface of the invention having immobilizedbiomolecules (e.g., target binding partners) is shown in FIG. 1A.

A representative dual-functional surface 5 is illustrated in FIG. 1B.Referring to FIG. 1B, a plurality of polymers 20 having carboxylic acidgroups are coupled to substrate 10 through a plurality of linkers 15.The polymers can form a monolayer on the surface of the substrate 10. Aportion of the carboxylic acid groups are used to form amide bonds withthe amino groups of target binding partners 25 to covalently couple thetarget binding partners 25 to the polymers 20. The coating of thepolymers 20 on the substrate 10 renders the surface 5 nonfouling whilethe immobilized target binding partners 25 provide binding affinity to atarget molecule.

As illustrated in FIG. 1B, the polymers can be attached to the surfacedirectly or through a plurality of linkers. In one embodiment, thesubstrate of the invention further comprises a monolayer (e.g.,self-assembled) covalently coupled to the surface, wherein the monolayercomprises a plurality of alkylene moieties, and wherein the polymers arecovalently coupled to the plurality of alkylene moieties.

The binding affinity of a target molecule toward to the surface resultsfrom the target binding partners immobilized on the surface. The targetbinding partner and the target molecule, each termed a binding pairmember, form a binding pair. Each binding pair member is a molecule thatspecifically binds the other member. In one embodiment, the targetbinding partner has affinity to a target molecule with K_(d) less thanabout 10⁻⁸.

A binding pair member can be any suitable molecule including, withoutlimitation, proteins, peptides, proteins, poly- or oligo-saccharides,glycoproteins, lipids and lipoproteins, and nucleic acids, as well assynthetic organic or inorganic molecules having a defined bioactivity,such as an antibiotic, anti-inflammatory agent, or a cell adhesionmediator.

Examples of proteins that can be immobilized on the surfaces of thepresent invention include ligand-binding proteins, lectins, hormones,receptors, and enzymes. Representative proteins include antibodies(monoclonal, polyclonal, chimeric, single-chain or other recombinantforms), their protein/peptide antigens, protein-peptide hormones,streptavidin, avidin, protein A, proteins G, growth factors and theirrespective receptors, DNA-binding proteins, cell membrane receptors,endosomal membrane receptors, nuclear membrane receptors, neuronreceptors, visual receptors, and muscle cell receptors. Representativeoligonucleotides that can be immobilized on the surfaces of the presentinvention include DNA (genomic or cDNA), RNA, antisense, ribozymes, andexternal guide sequences for RNAase P, and can range in size from shortoligonucleotide primers up to entire genes.

Other target binding partners that bind specifically to a targetcompound include poly- or oligosaccharides on glycoproteins that bind toreceptors, for example, the carbohydrate on the ligand for theinflammatory mediators P-selectin and E-selectin, and nucleic acidsequences that bind to complementary sequences, such as ribozymes,antisense, external guide sequences for RNAase P, and aptamers.

In one embodiment, the target binding partner is an antibody, and thetarget molecule is an antigen against the antibody. In this embodiment,the surface of the invention specifically binds to the antigen andresists non-specific protein adsorption. In one embodiment, the targetbinding partner is a protein capable of promoting cell adhesion, and thetarget molecule is a cell. In this embodiment, the surface of theinvention specifically binds to the cell and resists non-specificprotein adsorption and non-specific cell adhesion.

A variety of substrate surfaces can be rendered dual-functional usingthe materials and methods described herein. Representative surfaces thatcan be rendered dual-functional include metal and metal oxide surfaces,ceramic surfaces, synthetic and natural polymeric surfaces, glasssurfaces, fiber glass surface, silicon/silica surfaces, and carbon-basedmaterial surfaces. Representative natural polymeric surfaces includecollagen, fibrins, and other carbohydrate surfaces suitable for the useof tissue engineering. Representative carbon-based material surfacesinclude carbon fiber, nanotube, and bulky ball surfaces.

Suitable substrates include those substrates useful in medicaldiagnostic applications such as biosensors, bioprobes, and biomedicaldevices including in vivo devices; biomaterials and tissue engineeringapplications, such as membranes for bioprocesses or bioseparation,implantable devices, prosthetics, and tissue scaffolds; and drugdelivery applications, such as particles and nanoparticles.

In one embodiment, the present invention provides a SPR sensor on whichzwitterionic polymers were grafted to an SPR surface followed by theimmobilization of target binding partners to provide a dual-functionalsurface.

As noted above, suitable polymers useful for making the modifiedsurfaces of the invention include polymers derived from zwitterionicmonomers (as well as monomers that could be converted to zwitterionicmonomers, e.g., precursors of zwitterionic monomers). Zwitterionicmonomers are electronically neutral monomers that typically includeequal numbers of positive and negative charges (e.g., one of each). Inthe practice of the invention, suitable polymers include any polymerderived from zwitterionic monomers or precursors to zwitterionicmonomers that is capable of rendering a surface nonfouling and providingreactive functional groups on the surface for immobilization of a memberof a binding pair.

In certain embodiments, the polymers are covalently coupled to thesurface through a self-assembly monolayer, wherein the self-assemblymonolayer comprises a plurality of alkylene moieties.

In one embodiment, the polymer is a homopolymer prepared fromzwitterionic monomers and has the formula:

wherein B is selected from the group consisting of

wherein R is selected from the group consisting of hydrogen andsubstituted or unsubstituted alkyl; and E is selected from the groupconsisting of substituted or unsubstituted alkylene, —(CH₂)_(p)C(O)O—,and —(CH₂)_(p)C(O)NR²—, wherein p is an integer from 0 to 12, and R² isselected from the group consisting of hydrogen and substituted orunsubstituted alkyl;

L is a straight or branched alkylene group optionally including one ormore oxygen atoms;

P is selected from the group consisting of

wherein R³ and R⁴ are independently selected from the group consistingof hydrogen and substituted or unsubstituted alkyl group, R⁵ is selectedfrom the group consisting of substituted or unsubstituted alkylene,phenylene, and polyether groups, and

m is an integer from 1 to 7; and

x is an integer from 3 to 1000.

In one embodiment, the polymer is a poly(carboxybetaine). Suitablepoly(carboxybetaine)s can be prepared from one or more monomers selectedfrom the group consisting of carboxybetaine acrylates, carboxybetaineacrylamides, carboxybetaine vinyl compounds, carboxybetaine epoxides,and mixtures thereof. In one embodiment, the monomer is carboxybetainemethacrylate. Representative monomers for making carboxybetaine polymersuseful in the invention include carboxybetaine methacrylates, such as2-carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl) ethanaminium innersalt; carboxybetaine acrylates; carboxybetaine acrylamides;carboxybetaine vinyl compounds; carboxybetaine epoxides; and othercarboxybetaine compounds with hydroxyl, isocyanates, amino, orcarboxylic acid groups. In one embodiment, the polymer is apoly(carboxybetaine methacrylate) (poly(CBMA)).

The poly(carboxybetaine) can be prepared by polymerization methodsincluding atom transfer radical polymerization (ATRP), reversibleaddition fragmentation chain transfer (RAFT) polymerization, and freeradical polymerization. Any conventional radical initiators forpolymerization may be used.

In another embodiment, the polymer is a homopolymer that has a positivecharge in the polymer backbone and a pendant carboxylic acid group andhas the formula:

wherein R is selected from the group consisting of hydrogen andsubstituted or unsubstituted alkyl;

L₁ and L₂ are independently a straight or branched alkylene groupoptionally including one or more oxygen atoms; and

x is an integer from 3 to 1000.

A representative zwitterionic polymer 3 with a positively chargedbackbone can be obtained as shown in FIG. 2. Referring to FIG. 2,polymer 3 can be synthesized with aziridine as the starting material.N-methyl aziridine monomer is obtained by methylation of aziridine.Intermediate polymer 1 having a positively charged backbone is obtainedthrough a ring-open polymerization of N-methyl aziridine monomer.Quaternization of polymer 1 provides intermediate polymer 2 with methylesters as the pendant group. Hydrolysis of the methyl esters yields thedesired zwitterionic polymer 3.

In another embodiment, the polymer is a mixed charge copolymer and hasthe general formula:

wherein B₁ and B₂ are independently selected from the group consistingof

wherein R is selected from the group consisting of hydrogen andsubstituted or unsubstituted alkyl; and E is selected from the groupconsisting of substituted or unsubstituted alkylene, —(CH₂)_(p)C(O)O—,and —(CH₂)_(p)C(O)NR²—, wherein p is an integer from 0 to 12, and R² isselected from the group consisting of hydrogen and substituted orunsubstituted alkyl;

L is a straight or branched alkylene group optionally including one ormore oxygen atoms;

P₁ is a positively charged group;

P₂ is carboxylic acid group;

m is an integer from 3 to 1000; and

n is an integer from 3 to 1000.

In this embodiment, P₁ is nitrogen in an aromatic ring or NR₅R₆, whereinR₅ and R₆ are independently substituted or unsubstituted alkyl group.

The positively charged unit (P₁ containing unit) can be a derived from amonomer having a positively charged pendant group. Representativemonomers that can be used to derive the positively charged unit in thepolymers of the present invention include 2-(dimethylamino)ethylmethacrylate, 2-(diethylamino)ethyl methacrylate,[2-(methacryloyloxy)ethyl]trimethylammonium chloride, andN-acetylglucosamine.

The mixed charged copolymers or precursors thereof useful in the presentinvention can be synthesized directly on a substrate surface. The mixedcharged copolymers can be grafted onto a surface via atom transferradical polymerization (ATRP) from monomers having oppositely chargedgroups. The copolymers grafted on the surface can form substantiallyelectronically neutral polymer brushes having nonfouling properties.

In one embodiment, the negatively charged unit is derived from2-carboxyethyl acrylate (CA), and the positively charged unit is derivedfrom 2-(dimethylamino)ethyl methacrylate (DM). In one embodiment, thenegatively charged unit is derived from 2-carboxyethyl acrylate (CA),and the positively charged unit is derived from 2-(diethylamino)ethylmethacrylate (DE). In one embodiment, the negatively charged unit isderived from 2-carboxyethyl acrylate (CA), and the positively chargedunit is derived from [2-(methacryloyloxy)ethyl]trimethylammoniumchloride (TM). In one embodiment, the negatively charged unit is derivedfrom 2-carboxyethyl acrylate (CA), and the positively charged unit isderived from 2-aminoethyl methacrylate hydrochloride (NH2).

For any of the above surfaces, the plurality of polymers can form amonolayer on the surface.

In another aspect, the present invention provides methods for modifyinga surface that render the surface nonfouling and having affinity towarda target molecule.

In one embodiment, the method for modifying a surface, includescovalently coupling a plurality of target binding partners to aplurality of polymers that are attached (e.g., covalently coupled) to asurface of a substrate. The target binding partner has affinity to atarget molecule. The polymers comprise a plurality of carboxylic acidgroups and a plurality of positive charged groups, and wherein thepolymer is substantially electronically neutral. The polymers useful inthese methods include the polymers described in detail above with regardto the surfaces of the invention. In one embodiment, the polymer is apoly(carboxybetaine).

In certain embodiments, covalently coupling the plurality of targetbinding partners to the plurality of polymers includes forming an amidelinkage between the polymers and the target binding partners. In oneembodiment, covalently coupling the plurality of target binding partnersto the plurality of polymers includes converting a portion of carboxylicacid groups to activated esters and reacting the activated esters withtarget binding partners having amino groups. For polymers that have beenmodified to include available amino groups, covalently coupling theplurality of target binding partners to the plurality of polymersincludes converting a portion of carboxylic acid groups of the targetbinding partners to activated esters and reacting the activated esterswith the polymers of the surface having available amino groups.

In one embodiment, the activated esters are N-hydroxysuccinimide estersprepared as described below.

Suitable target binding partners include those described in detail abovewith regard to the surfaces of the invention.

The invention provides substrates, as described above, having modifiedsurfaces prepared by the methods of the invention.

As noted above, the carboxylic acid groups of the polymers may beactivated by forming activated intermediate groups for further couplingwith the amino groups of the biomolecule. Any activated form ofcarboxylic acid groups may be used in the present invention. Therepresentative activated intermediate groups include carbodiimides,carbonyldiimidazoles, uranium salts, isothiocyanates, isocyanates, acylazides, N-succinimidyl esters (NHS esters), sulfonyl chloride,aldehydes, epoxides arylating groups, imido esters, and anhydrides. Inone embodiment, O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TSTU) is used to convert the carboxylic acid groupson the polymers to activated uronium salts. In another embodiment,carbodiimides and N-hydroxysuccinimide (EDC/NHS coupling chemistry) areused to convert the carboxylic acid groups on the polymers to activatedN-hydroxysuccinimide esters.

In one embodiment, the carboxylic acid groups were converted toactivated NHS esters by treating the carboxylic acid groups with N-bromosuccinimide (NHS) and N-ethyl-N′-(3-diethylaminopropyl) carbodiimide(EDC) under acidic conditions (e.g., pH 2.5 to 6). The activated NHSesters were coupled with the amino groups of the target binding partnerforming amide bonds under basic conditions (e.g., pH 7.4 to 11).

The polymer may be coupled to the surface through alkylene linkers. Thegrafting of the polymers onto the surface of the substrate through thealkylene linkers may be via any conventional polymerization method suchas atom transfer radical polymerization (ATRP), reversible additionfragmentation chain transfer (RAFT) polymerization, and free radicalpolymerization.

SAMs on substrate surfaces are an excellent platform for surfacepolymerization. In one embodiment, a radical initiator-terminatedself-assembly monolayer (SAM) comprising the alkylene linkers can beformed onto the substrate surface, and the polymers are grafted onto thesurface through the radical initiator-terminated self-assemblymonolayer. In this method, the substrate surface can be coated with theSAMs terminated with radical initiator, wherein the radical initiatorsare tethered to the surface through alkylene linkers. The alkylenelinkers can be any substituted or unsubstituted alkylene. In oneembodiment, the alkylene linker is a C2-C30 alkylene. Polymers are thenformed onto the SAMs to provide a layer of nonfouling polymeric coatingon the substrate surface. Atom transfer radical polymerization isinitiated by the radical initiator at the SAMs terminus.

In one embodiment, a hydroxyl-terminated monolayer having alkylenelinkers can be formed onto the substrate surface, which is subsequentlyconverted to a radical initiator terminated monolayer.

A representative dual-functional surface comprising poly(carboxybetainemethacrylate) is described in Example 1 and its preparation illustratedin FIG. 3.

The representative nonfouling surface was prepared by graftingpoly(carboxybetaine methacrylate) onto a substrate surface covered withinitiators via the surface-initiated ATRP method (FIG. 3).ω-Mercaptoundecyl bromoisobutyrate was synthesized by reactingbromoisobutyryl bromide and mercaptoundecanol. The initiators wereimmobilized on a gold substrate via self-assembly by soaking the goldsubstrate in a solution containing w-mercaptoundecyl bromoisobutyrate.One of the CBMA monomers,2-carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl) ethanaminium innersalt, was synthesized by reacting 2-(N,N′-dimethylamino)ethylmethacrylate with β-propiolactone. The CBMA monomers were grafted fromradical initiator-terminated SAMs via ATRP. CuBr and 2,2′-bipyridine(BPY) were used as a catalyst and a ligand, respectively. The reactionwas kept under mild conditions at room temperature in a mixed solvent ofmethanol and water. After a typical ATRP polymerization, homogenouscarboxybetaine polymer brushes were grafted to the surface. Thethickness of the polymer layer was around 10-15 nm as measured byellipsometry.

Protein adsorption was measured with a surface plasmon resonance sensor(SPR) based on wavelength interrogation. Protein adsorption is definedas the wavelength shift before protein injection and after buffer wash.The wavelength shift after protein injection is mainly due to the changein the bulk refractive index. The adsorption of three proteins withdifferent sizes and isoelectric points (pI)—human fibrinogen (340 kD,pI=5.5), lysozyme (14 kD, pI=12), and hCG (37 kD, pI=4.5)—onpoly(CBMA)-grafted surfaces was shown to decrease to <0.3 ng/cm² (or awavelength shift of <0.02 nm, the detection limit of the SPR sensor), asshown in FIG. 4. This level of adsorption is the same as that found forwell-packed OEG SAMs or surfaces covered with poly(CBMA) viasurface-initiated ATRP. Thus, poly(CBMA)-grafted surfaces are highlyresistant to non-specific protein adsorption.

Following monolayer formation on the surface, a representative targetbinding partner, a monoclonal antibody (mAb) to human chorionicgonadotropin (hCG) was covalently immobilized onto the surface coveredwith poly(CBMA) brushes by reacting the amines of the mAb to thecarboxyl groups of the poly(CBMA) via EDC/NHS coupling chemistry asshown in FIG. 3.

After the immobilization, unreacted NHS was removed with ethanolamine.The SPR response after an injection of hCG shows that the adsorbedamount of protein is much greater on the poly(CBMA)-grafted surface withimmobilized anti-hCG than on the control poly(CBMA)-grafted surfaceswithout the immobilized mAb. FIG. 5 shows the protein adsorption on thepoly(CBMA)-grafted surface with immobilized anti-hCG when the surface istreated with PBS solution containing 1 mg/mL fibrinogen, 1 mg/mLlysozyme, and 20 μg/mL hCG for 22 min. FIG. 6 shows the proteinadsorption on the poly(CBMA)-grafted surface with immobilized anti-hCGwhen the surface is treated with PBS solution containing 1 mg/mLfibrinogen, 1 mg/mL lysozyme, and 40 μg/mL hCG for 22 min. Thewavelength shift of the SPR spectra is 1.6 nm, corresponding to hCGadsorption of about 24 ng/cm². At the same time, the adsorption of bothlysozyme and fibrinogen remains the same on both surfaces (less than 0.3ng/cm²), indicating that the surface is still nonfouling after anti-hCGimmobilization. Thus, the poly(CBMA)-grafted surface with an immobilizedanti-hCG represents a dual-functional surface with both resistance tonon-specific protein adsorption and binding to a specific protein.

The activation of carboxylic acid groups of poly(carboxybetaine) on thesurface is important for the subsequent immobilization of target bindingpartners. To achieve high protein immobilization level, the conditionsfor the activation of carboxylic acid groups and the immobilization ofthe target binding partners need to be optimized.

As shown in Example 2, the optimal activation of carboxylic acid groupsof the polymers using NHS/EDC method (the greatest amount of NHS esters)was achieved in situ using water/HCl, pH 3.3 as a solvent. The surfacecoated with poly(carboxybetaine methylacrylate) was activated byinjection of freshly prepared solution of NHS (0.05M) and EDC (0.2M) at30° C. Two different antibodies, monoclonal antibody against activatedcell leukocyte adhesion molecule (anti-ALCAM) and polyclonal antibodyagainst Salmonella (anti-Salm), were immobilized into different spots ofthe activated CBAA surface under various pH conditions with antibodyconcentration of 50 ug/mL. Then, the non-covalently bound ligands wereremoved from the surface.

The effect of pH on antibody immobilization is shown in FIG. 7. It isapparent from FIG. 7 that higher pH of immobilization conditioncorresponds to higher amount of immobilized protein. A typical responseto immobilization of antibody at pH 8.0 is shown in FIG. 8. FIG. 9compares the detection of ALCAM on an anti-ALCAM immobilized surface anda reference surface with immobilized anti-Salm in 50% plasma.

The activation of the carboxylic acid groups of poly(carboxybetainemethylacrylate) may be achieved by treating the carboxylic acid groupswith TSTU as described in Example 3. A custom-built four-channelspectroscopic surface plasmon resonance sensor was used to monitorpoly(CBAA) surface functionalization.

Poly(CBAA) chips were first prepared in the form of Poly(CBAA) estersvia ATRP method. The poly(CBAA)-ester surface is then hydrolyzed toachieve zwitterionic nonfouling poly(CBAA) surface. A sensor chipsurface with grafted poly(CBAA) esters was washed with MilliQ water,dried with filtered air and mounted into the SPR sensor. The hydrolysiswas performed in situ by flowing with 0.1M NaOH, pH 12 for 2 hours alongthe poly(CBAA) surface. The effectiveness of hydrolysis was monitored byinjection of highly surface-sticking protein (fibrinogen) withconcentration of 1 mg/mL in phosphate buffered saline (PBS), pH 7.4. Itwas found that 2 hours was sufficient time to fully hydrolyze thepoly(CBAA) ester surface.

The zwitterionic poly(CBAA) surface was immersed in solution of TSTU (5mg/mL) in DMF for 2 hours. After that, the surface was washed withethanol and dried with filtered air. The chip was immediately mountedinto the SPR sensor. The model system of two antibodies was used todemonstrate functionalization of the surface-grafted poly(CBAA).Antibody against activated cell leukocyte adhesion molecule (anti-ALCAM)was immobilized into the measuring channel and antibody againstSalmonella (anti-Salm) was immobilized into the reference channel.Immobilization was performed in situ in 10 mM sodium acetate buffer, pH5.0. Antibody concentration was consecutively 10 ug/mL and 50 ug/mL,incubation time was 15 minutes.

FIG. 10 shows sensor response to immobilize ALCAM and Salmonellaantibodies on the surface of a poly(CBAA) sensor activated with TSTU.Running buffer was 10 mM sodium acetate, pH 5.0 with the concentrationof antibody as 10 ug/mL and 50 ug/mL, consecutively. FIG. 11 shows thedetection of ALCAM using the anti-ALCAM immobilized surface and lowbackground noise (or low non-specific binding) using a non-targetedantigen, fibrinogen. Residual NETS-esters were transformed back tocarboxylate groups by flowing 10 mM phosphate buffer, 150 mM NaCl, pH8.5 for 5 hours. Fibrinogen with concentration of 1 mg/mL was injectedinto reference channel to check for the effectiveness of hydrolysis andsuper nonfouling properties (FIG. 11A). Detection of ALCAM antigen wasperformed then in PBS buffer. A solution of ALCAM with concentration of100 ng/mL was injected over the measuring and reference channel for 10minutes. FIG. 11B shows sensor response to ALCAM binding in themeasuring (anti-ALCAM) and reference (anti-Salm) channels. ALCAM antigenwas bound specifically to the anti-ALCAM surface. These resultsdemonstrate that deactivation process was successful and functionalizedpoly(CBAA) surface kept its super nonfouling properties while thebiological activity of anti-ALCAM was preserved.

In other aspects, the invention provides materials having target bindingpartners immobilized thereto that are useful for rendering surfacesnonfouling. These materials are polymeric materials that can be used tocoat surfaces. These materials can be used independently of substratesurfaces. The materials can be formed into particles and used inparticulate form. Representative materials of the invention includecrosslinked polymers (e.g., hydrogels) having target binding partnersimmobilized thereto and block copolymers (e.g., microparticles andnanoparticles) having target binding partners immobilized thereto.

In one embodiment, the present invention provides a crosslinked polymer(e.g., hydrogel) having a plurality of target binding partnerscovalently coupled thereto.

In one embodiment, the crosslinked polymer has a plurality of targetbinding partners covalently coupled thereto. The crosslinked polymershave a plurality of carboxylic acid groups and a plurality of positivecharged groups, and are substantially electronically neutral. The targetbinding partner has affinity toward a target molecule.

The polymers making up the crosslinked polymers include those describedabove in regard to the surfaces of the invention.

In one embodiment, the crosslinked polymer is a crosslinkedpoly(carboxybetaine) hydrogel. The carboxybetaine can be prepare fromone or more monomers selected from the group consisting ofcarboxybetaine acrylates, carboxybetaine acrylamides, carboxybetainevinyl compounds, carboxybetaine epoxides, and mixture thereof. In oneembodiment, the monomer is carboxybetaine methacrylate.

Representative crosslinked polymers can be prepared from positivelycharged compounds including aminoethyl methacrylate hydrochloride (NH2),(2-(dimethylamino)ethyl methacrylate (DM), 2-(diethylamino) ethylmethacrylate (DE), and 2-(methacryloyloxy)ethyl trimethylammoniumchloride (TM), and negatively charged compounds including 2-carboxyethylacrylate (CA).

In one embodiment, the crosslinked polymer NH2/CA has the negativelycharged unit derived from 2-carboxyethyl acrylate (CA) and thepositively charged unit derived from 2-aminoethyl methacrylatehydrochloride (NH2). In one embodiment, the crosslinked polymer DM/CAhas the negatively charged unit derived from 2-carboxyethyl acrylate(CA) and the positively charged unit derived from 2-(dimethylamino)ethylmethacrylate (DM). In one embodiment, the crosslinked polymer DE/CA hasthe negatively charged unit derived from 2-carboxyethyl acrylate (CA)and the positively charged unit derived from 2-(diethylamino)ethylmethacrylate (DE). In one embodiment, the crosslinked polymer TM/CA hasthe negatively charged unit derived from 2-carboxyethyl acrylate (CA)and the positively charged unit derived from[2-(methacryloyloxy)ethyl]trimethylammonium chloride (TM).

Immobilization of the target binding partner to the crosslinked polymerscan be carried out as described above for the surfaces of the invention.The target binding partner can be immobilized onto the polymer by, forexample, reacting the amino groups of the protein to the carboxylic acidgroups available on the surface of crosslinked polymer. The targetbinding partners include those described above in regard to the surfacesof the invention.

The present invention provides methods for making crosslinked polymershaving a plurality of target binding partners covalently coupled to thepolymers.

In one embodiment, the method for making a surface-modified crosslinkedpolymer includes:

(a) copolymerizing one or more monomers and a crosslinking agent toprovide a crosslinked polymer, wherein the crosslinked polymer comprisescrosslinked polymers having a plurality of carboxylic acid groups and aplurality of positive charged groups, wherein the crosslinked polymersare substantially electronically neutral; and

(b) covalently coupling a plurality of target binding partners to thecrosslinked polymer.

In the method, covalently coupling a plurality of target bindingpartners to the crosslinked polymer can be carried out using thematerials and methods described above for the preparation of thesurfaces of the invention.

Suitable crosslinking agents include those commonly used in vinyl-typepolymer crosslinking. In one embodiment, the crosslinking agent istetraethylene glycol dimethacrylate.

A representative crosslinked polymer hydrogel of the invention, acrosslinked poly(CBMA) hydrogel, was prepared as described in Example 4.The transparent hydrogel was prepared by adding carboxybetainemethacrylate monomer into tetraethylene glycol dimethacrylate (TEGDMA)followed by free radical polymerization initiated by sodiummetabisulfite and ammonium persulfate. After polymerization, the gel wasprepared according to the well-established procedure known in the art toremove residual chemicals. The poly(CBMA) hydrogel was found to benoncytotoxic and contained less than 0.06 units (EU)/mL of endotoxinusing a Limulus amebocyte lysate (LAL) endotoxin assay kit (CambrexBioscience, Walkersville, Md.). The hydrogel was punched into disks.

The crosslinked poly(CBMA) hydrogel was then evaluated for its dualfunctionality. The poly(CBMA)-based hydrogel was modified by EDC/NHSactivation followed by fibronectin conjugation to provide a modifiedhydrogel decorated with immobilized fibronectin. The fibronectin is aprotein capable of promoting cell adhesion. Following proteinconjugation, bovine aortic endothelial cells (BAECs) were cultured onboth fibronectin-modified hydrogel disks and the hydrogel disks withoutEDC/NHS activation. Cell morphology was observed between 2 h and 3 daysof cultivation. While there were no adhered cells on the poly(CBMA)hydrogel disks without EDC/NHS activation as shown in FIG. 12A, BAECswere observed on the surfaces that were activated with EDC/NHS beforethe fibronectin conjugation (FIG. 12B). These results show that thepoly(CBMA) hydrogel itself is highly resistant to cell adhesion and canbe readily immobilized with proteins or other ligands.

In another embodiment, the invention provides block copolymers having aplurality of target binding partners covalently coupled thereto. Theblock polymers have a first hydrophilic block and a second hydrophobicblock. The hydrophilic block includes a plurality of carboxylic acidgroups and a plurality of positive charged groups, and is substantiallyelectronically neutral. Covalently coupling a plurality of targetbinding partners to the block copolymers can be carried out using thematerials and methods described above for the preparation of thesurfaces and crosslinked polymers of the invention. These blockcopolymers can be advantageously used in the form of microparticles andnanoparticles.

The diblock copolymers have the general formula [hydrophobicmonomer]₁-block-[hydrophilic monomer]_(m) copolymer, where 1 is aninteger from 10-30 and m is an integer from 10 to 100. In oneembodiment, the hydrophilic block is a poly(carboxybetaine).

In one embodiment, well-defined diblock copolymers containingcarboxybetaine moieties, such as poly(CBMA), with a hydrophobic moiety,such as poly(propylene oxide) (PPO), are adsorbed onto surfaces coatedwith alkyl-terminated SAMs, such as methyl (CH₃)-terminated SAMs. Forthis embodiment, the hydrophobic polymer segment binds to thehydrophobic surface and the hydrophilic sulfobetaine moiety is exposedto, for example, an aqueous solution in contact with the surface.

The diblock copolymers of the invention can be composed of a variety ofcarboxybetaine-based hydrophilic portions having varied length and anysuitable hydrophobic portion with varied length, and the diblockcopolymers can be prepared by any suitable methods of polymerization.

Representative monomers for making carboxybetaine polymers useful in theinvention include carboxybetaine methacrylates, such as2-carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl) ethanaminium innersalt; carboxybetaine acrylates; carboxybetaine acrylamides;carboxybetaine vinyl compounds; carboxybetaine epoxides; and othercarboxybetaine compounds with hydroxyl, isocyanates, amino, orcarboxylic groups. Any hydrophobic polymer chains could be used as thehydrophobic moiety for the copolymer of the invention. Representativehydrophobic moieties include poly(propylene oxide) (PPO),polymethacrylates, polyacrylates, polyacrylamides, polyesters,polyethers, polyurethanes, and polyamides.

In one embodiment, two block copolymers can be used together. In oneembodiment, the first diblock copolymer comprises a [hydrophobicmonomer]₁-block-[hydrophilic monomer]_(m) copolymer. In one embodiment,the first diblock copolymer comprises a [propyleneoxide]₁-block-[carboxybetaine methacrylate]_(m) copolymer. In oneembodiment, the second diblock copolymer comprises a [hydrophobicmonomer]₁-block-[hydrophilic monomer]_(n) copolymer. In one embodiment,the second diblock copolymer comprises a [propyleneoxide]₁-block[carboxybetaine methacrylate]_(n) copolymer. For thesepolymers 1 is an integer from 10-30, m is an integer from 10-100, n isan integer from 10-50, and m is greater than n.

The materials and methods of the invention can be used advantageously ina variety of applications. In one embodiment, the materials and methodsof the invention can be used to prepare protein arrays for performingprotein assays.

Non-specific adsorption of proteins is routinely observed on traditionalprotein arrays, leading to high background levels. The problem isespecially noticeable when complex protein solutions are used, such asblood, tissue or cell lysate. Non-specific adsorption of non-targetproteins compromises the specificity of any sensor surface, and makesthe results less reliable. The advantages of using a surface platformfor protein arrays made from the materials and by the methods of theinvention include (a) excellent nonfouling characteristics in bloodserum and plasma, (b) abundant functional groups for proteinimmobilization, and (c) activated surfaces for protein immobilizationcan be automatically de-activated back to a superlow fouling background.None of the existing surface chemistries have all of these uniqueproperties together.

FIG. 13A is a schematic illustration of an array (a pCBMA-based surfaceplatform for protein arrays) using the materials and the methods of theinvention. A gold coated surface or a glass slide is grafted with pCBMAvia ATRP. The surface (i.e., the carboxylic acid moiety of thezwitterionic carboxybetaine group) can be activated using the two-stepNHS/EDC chemistry described above. Proteins are then spotted directlyonto the surface using a spotter. The chip is automatically de-activatedwith buffer where the activated carboxylic acid moiety returns to itsoriginal carboxylate ion, leading to a nonfouling background of Pcbma.FIG. 13B is a schematic illustration of conventional mixed COOH/OH OEGSAM surface platform for protein arrays and the only final stage afterprotein immobilization is shown. Nonspecific protein binding fromcomplex medium like blood serum or plasma is very sensitive to theexistence of the excess or unreacted COOH groups on the protein arraysurface. The inherent problem for conventional surface chemistries isthat they contain two separate functional and nonfouling groups.

The preparation of a representative protein array and its use inperforming protein assays is described in Example 5.

The following is a listing of abbreviations used herein.

Abbreviations

AIBN Azobisisobutylonitrile

ALCAM Activated cell leukocyte adhesion molecule

ATRP atom transfer radical polymerization

BAEC Bovine aortic endothelial cell

C12 Lauryl methacrylate

CA 2-Carboxyethyl acrylate

CBMA Carboxybetaine methylacrylate

DE 2-(Diethylamino) ethyl methacrylate

DM 2-(Dimethylamino)ethyl methacrylate

DMF Dimethylformamide

E Glutamic Acid

EDC N-Ethyl-N′-(3 -dietyhlaminopropyl)carbodiimide

EDT 1,2-ethanedithiol

ELISA Enzyme-linked Immunosorbent Assay

GL Ethyl glycolate methacrylate

hCG human chorionic gonadotropin

IB Isobutyl methacrylate

Me Methyl methacrylate

NH₂ 2-Aminoethyl methacrylate hydrochloride

NHS N-bromo succinimide

NHS ester N-succinimidyl ester

PEG Poly(ethylene glycol) methacrylate

RAFT reversible addition fragmentation chain transfer

SAM Self-assembly monolayer

SP 3-Sulfopropyl methacrylate potassium salt

SPR Surface plasmon resonance

TCPS Tissue culture polystyrene

TFA Trifluoroacetic acid

TFE 2,2,2-Trifluoroethyl methacrylate

TM [2-(Methacryloyloxy) ethyl]trimethylammonium chloride

TSTU N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uranium tetrafluoroborate

The following examples are provided for the purpose of illustrating, notlimiting, the invention.

EXAMPLES

Materials. Human plasma fibrinogen and chicken egg white lysozyme werepurchased from Sigma-Aldrich (Milwaukee, Wis.). Human plasma fibronectinwas purchased from Chemicon International (Temecula, Calif.). Humanchorionic gonadotropin (hCG) and its monoclonal mouse antibody (mAb;isotype IgGl) were purchased from Scripps Laboratories (San Diego,Calif.). 2-(N,N′-dimethylamino)ethyl methacrylate (DMAEM, 98%),β-propiolactone (95%), copper (1) bromide (99.999%), bromoisobutyrylbromide (98%), β-mercapto-1-undecanol (97%), 2,2′-bipyridine (BPY 99%),tetrahydrofuran (THF, HPLC grade), NHS, and EDC, were purchased fromSigma-Aldrich (Milwaukee, Wis.). Phosphate-buffered saline (PBS: 0.01 Mphosphate, 0.138 M sodium chloride, 0.0027 M potassium chloride, pH 7.4)was purchased from Sigma Chemical Co. Ethanol (absolute 200 proof) waspurchased from AAPER Alcohol and Chemical Co. Water used in experimentswas purified using a Millipore water purification system with a minimumresistivity of 18.0 MΩ•cm. THF for reactions and washings was dried bysodium before use.

Example 1 Representative Dual-Functional Surface Having NonfoulingPoly(CBMA) Coating

Human plasma fibrinogen and chicken egg white lysozyme were purchasedfrom Sigma-Aldrich (Milwaukee, Wis.). Human plasma fibronectin waspurchased from Chemicon International (Temecula, Calif.). Humanchorionic gonadotropin (hCG) and its monoclonal mouse antibody (isotypeIgGl) were purchased from Scripps Laboratories (San Diego, Calif.).2-(N,N′-dimethylamino)ethyl methacrylate (DMAEM, 98%), β-propiolactone(95%), copper (I) bromide (99.999%), bromoisobutyryl bromide (98%),11-mercapto-1-undecanol (97%), 2,2′-bipyridine (BPY 99%) andtetrahydrofuran (THF HPLC grade), N-hydroxysuccinimide (NHS) and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), were purchasedfrom Sigma-Aldrich (Milwaukee, Wis.). Phosphate buffer saline (PBS, 0.01M phosphate, 0.138 M sodium chloride, 0.0027 M potassium chloride, pH7.4) were purchased from Sigma Chemical Co. Ethanol (absolute 200 proof)was purchased from AAPER Alcohol and Chemical Co. Water used inexperiments was purified using a Millipore water purification systemwith a minimum resistivity of 18.0 MΩcm. THF for reactions and washingswere dried by sodium before use.

A SPR glass chip was coated with an adhesion-promoting chromium layer(thickness 2 nm) and a surface plasmon active gold layer (48 nm) byelectron beam evaporation under vacuum. Before SAM preparation, thesubstrates were treated as reported method. The initiator,w-mercaptoundecyl bromoisobutyrate, was synthesized through reaction ofbromoisobutyryl bromide and 11-mercapto-1-undecanol using a methodpublished previously.

CBMA was synthesized by reaction of 2-(N,N′-dimethylamino)ethylmethacrylate with β-propiolactone. The ATRP surface initiatedpolymerization was reported in our previous papers.

Mercaptoundecyl bromoisobutyrate was synthesized through the reaction ofbromoisobutyryl bromide and 11-mercapto-1-undecanol using a methodpublished previously. 1H NMR (300 MHz, CDCl₃): 4.15 (t, J=6.9 Hz, 2H,OCH₂), 2.51 (q, J=7.5 Hz, 2H, SCH₂), 1.92 (s, 6H, CH₃), 1.57-1.72 (m,4H, CH₂), and 1.24-1.40 (m, 16H, CH₂).

CBMA synthesis. A carboxybetaine methacrylate (CBMA) monomer,2-carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl)ethanaminium innersalt, was synthesized by reaction of 2-(N,N′-dimethylamino)ethylmethacrylate (DMAEM, 98%) with β-propiolactone (95%). 0.87 g (12 mmol)β-Propiolactone in 10 mL dried acetone was added dropwise to a solutionof 1.57 g (10 mmol) DMAEM dissolved in 50 mL dried acetone. The reactionwas stirred under nitrogen protection at 15° C. for about 5 hours. Thewhite precipitate was washed with 50 mL dried acetone and 100 mL driedether. The product was dried under reduced pressure to get CBMA monomer.The monomer was kept at 2-8° C. before the polymerization. Yield: 91%.

Surface initiated polymerization on a SPR sensor. SPR glass chips werecoated with an adhesion-promoting chromium layer (2 nm) and a surfaceplasmon active gold layer (48 nm) by electron beam evaporation undervacuum. Before SAM preparation, the substrates were washed with pureethanol, cleaned under UV light, and washed with water and pure ethanol.The initiator SAMs were formed by soaking gold-coated substrates in apure ethanol solution of 1 mM ω-mercaptoundecyl bromoisobutyrate at roomtemperature for 24 hours. Before the polymerization, the substrates wererinsed with pure ethanol, followed by THF and dried in a stream ofnitrogen.

CuBr and the substrate with immobilized initiators were placed in areaction tube in a dry box under nitrogen protection. The tube sealedwith rubber septum stoppers was taken out. Degassed solution (pure waterand methanol in a 1:1 volume ratio) with CBMA and BPY was thentransferred to the tube using syringe under nitrogen protection. Afterthe reaction, the substrate was removed and rinsed with ethanol andwater, and the samples were kept in water overnight. Rinsing with PBSbuffer is also applied to remove unbound polymers before testing. For atypical polymerization, the substrate was reacted with 7.5 mmol CBMA, 2mmol BPY and 1 mmol CuBr in 25 mL CH₃OH/H₂O (1:1 volume ratio) for 1hour under nitrogen protection. After a typical ATRP polymerization,homogenous carboxybetaine polymer brushes were grafted on the goldsurface of a SPR sensor. The thickness of the polymer layer is around10-15 nm measured by ellipsometry.

SPR analysis and protein adsorption. Protein adsorption was measuredwith a custom-built surface plasmon resonance (SPR) sensor, which isbased on wavelength interrogation. A SPR chip (32×18×2 mm) was attachedto the base of the prism, and optical contact was established usingrefractive index matching fluid (Cargille). A four-channel flow cell(contact area: 17×3 mm each) with four independent parallel flowchannels was used to contain liquid sample during experiments. Aperistaltic pump (Ismatec) was utilized to deliver liquid sample to thefour channels of the flow cell. A fibrinogen solution of 1.0 mg/mL inPBS was flowed over the sensor surface at a flow rate of 0.05 mL/min. ASPR detector was used to monitor protein-surface interactions in realtime. Wavelength shift was used to measure the change in surfaceconcentration (or mass per unit area).

The smallest change in the signal that can be measured by the SPR sensorwas 0.02 nm or lower (i.e., 10 times the standard deviation of baselinenoise, which is 0.002 nm for the custom-built SPR sensor used). Fornormal molecules, at an operating wavelength of around 800 nm, a 1 minSPR wavelength shift corresponds to an adsorption of ˜15 ng/cm² on thesurface. For each surface, at least three samples were measured forprotein adsorption.

Ellipsometry. Ellipsometry was performed using a spectroscopicellipsometer (Sentech SE-850, GmbH). Sample preparation is the same asin XPS experiments. Five separate spots were measured at three differentangles of incidence (50, 60 and 70 degrees) in the visible region. Thesame batch of gold-coated chips was cleaned by UV-ozone cleaner for 20min, washed with ethanol and Millipore water, and dried with nitrogen.The bare gold-coated chips were used as a reference. The thicknesses offilms studied were determined using the Cauchy layer model with anassumed refractive index of 1.45.

Anti-hCG Immobilization on Poly(CBMA)-Grafted SPR Sensors. Themonoclonal mouse anti-hCG was immobilized onto the surface of thepolymer brushes by reacting amines of the antibody to the carboxylgroups of the carboxyl groups of the poly(CBMA) using EDC/NHS couplingchemistry. The poly(CBMA)-grafted SPR sensor surface was activated byincubating the SPR chip in a freshly prepared solution containing 2mg/mL NHS and 2 mg/mL EDC in a mixed solvent of dioxane/water (v/v 14:1)for 1 h at room temperature. The substrate was then removed from thesolution, rinsed with 18.2 mωcm DI water, and dried with a flow ofnitrogen. The anti-hCG mAb was linked to the activated surface byputting a 10 μL drop of 2 mg/mL anti-hCG in PBS onto the surface,covering the surface with a glass cover slip, and then incubating themAb with the activated surface for approximately 24 h at 4° C. in ahumid environment. The antibody functionalized substrate was washed with18.2 MΩcm DI water and excess activated sites were subsequently blockedby 1 M ethanolamine (pH 8.5) for 10 min remove any unreacted NHS.

Example 2 Optimization of Target Binding Partner Immobilization for aRepresentative Poly(CBAA) Coated Surface

Optimal activation of carboxylates using NHS/EDC method was achieved insitu using water/HCl, pH 3.3 as a solvent. Briefly, the polymer surfacewas activated by injection of freshly prepared solution of NHS (0.05M)and EDC (0.2M) for 10 minutes at temperature of 30° C. Two differentantibodies, in particular monoclonal antibody against activated cellleukocyte adhesion molecule (anti-ALCAM) and polyclonal antibody againstSalmonella (anti-Salm) were immobilized into different spots of theactivated poly(CBAA) coated surface under various pH conditions withantibody concentration of 50 ug/mL. The flow rate was 50 ul/min, T=30Cand incubation time was about 15 minutes. After that, the surface wastreated with 10 mM PB, 0.75M NaCl, pH 8.8 (PBNa) to remove allnon-covalently bound ligands. Effect of pH of running buffer on antibodyimmobilization is shown in FIG. 7. A typical response to immobilizationof antibody at pH 8.0 is shown in FIG. 8. FIG. 9 shows the detection ofALCAM on an anti-ALCAM immobilized surface and a reference surface withimmobilized anti-Salm in 50% plasma. CBAA monomer synthesis andpoly(CBAA) surface grafting are similar to those for CBMA as describedin Example 1 above.

Example 3 Representative Sensors with Dual-Functional Surface Coatedwith Poly(CBAA) and Activated with TSTU

A SPR sensor with a dual-functional surface based on nonfoulingpoly(CBAA) coating functionalized using the TSTU method was accomplishedin this example. A custom-built four-channel spectroscopic surfaceplasmon resonance sensor was used to monitor poly(CBAA) surfacefunctionalization.

Poly(CBAA) chips were first prepared in the form of Poly(CBAA) estersvia ATRP method. The poly(CBAA)-ester surface is then hydrolyzed toachieve zwitterionic nonfouling poly(CBAA) surface. A sensor chipsurface with grafted poly(CBAA) esters was washed with MilliQ water,dried with filtered air and mounted into the SPR sensor. The hydrolysiswas performed in situ by flowing with 0.1M NaOH, pH 12 for 2 hours alongthe poly(CBAA) surface. The effectiveness of hydrolysis was checked byinjection of highly surface-sticking protein (fibrinogen) withconcentration of 1 mg/mL in phosphate buffered saline (PBS), pH 7.4. Itwas found that 2 hours was sufficient time to fully hydrolyze thepoly(CBAA) ester surface.

The zwitterionic poly(CBAA) surface was immersed in solution of TSTU (5mg/mL) in DMF for 2 hours. After that, the surface was washed withethanol and dried with filtered air. The chip was immediately mountedinto the SPR sensor. The model system of two antibodies was used todemonstrate functionalization of the surface-grafted poly(CBAA).Antibody against activated cell leukocyte adhesion molecule (anti-ALCAM)was immobilized into the measuring channel and antibody againstSalmonella (anti-Salm) was immobilized into the reference channel.Immobilization was performed in situ in 10 mM sodium acetate buffer, pH5.0. Antibody concentration was consecutively 10 ug/mL and 50 ug/mL,incubation time was 15 minutes.

FIG. 10 shows sensor response to immobilize ALCAM and Salmonellaantibodies on the surface of a poly(CBAA) sensor twice (with 10 and 50ug/ml, respectively) that was activated with TSTU method. FIG. 11 showsthe detection of ALCAM using the anti-ALCAM immobilized surface and lowbackground noise (or low non-specific binding) using a non-targetedantigen (i.e., fibrinogen in this case). Residual NHS-esters weretransformed back to carboxylate groups by flowing 10 mM phosphatebuffer, 150 mM NaCl, pH 8.5 for 5 hours. Fibrinogen with concentrationof 1 mg/mL was injected into reference channel to check for theeffectiveness of hydrolysis and super nonfouling properties (FIG. 11A).Detection of ALCAM antigen was performed then in PBS buffer. Solution ofALCAM with concentration of 100 ng/mL was injected over the measuringand reference channel for 10 minutes. FIG. 11B shows sensor response toALCAM binding in the measuring (anti-ALCAM) and reference (anti-Salm)channels. ALCAM antigen was bound specifically to the anti-ALCAMsurface. These results suggest that deactivation process was successfuland functionalized poly(CBAA) surface kept its super nonfoulingproperties as well as the biological activity of anti-ALCAM waspreserved.

Example 4 Representative Carboxybetaine Hydrogel Preparation

In this example, a poly(CBMA) hydrogel and its resistance to proteinadsorption and cell adhesion is described. A CBMA hydrogel was preparedby adding 2.7 M CBMA monomer into tetraethylene glycol dimethacrylate(TEGDMA) (5.9 mol %) and through free radical polymerization initiatedby sodium metabisulfite (1.2 mol %) and ammonium persulfate(2.6 mol %)in a mixed solution (ethylene glycol/ethanol/H₂O=3:1:1 volume ratio).The reaction was carried out between a pair of glass substrates,separated with a PTFE spacer of 0.4 mm at 37° C. for 12 h. Afterpolymerization, the gel was immersed in a large amount of DI water forthree days and water was changed every day to remove residual chemicals.The gel was then equilibrated in sterilized PBS solution, which waschanged every day for another two days. Hydrogels were punched intodisks with a diameter of 5 mm and stored in sterilized buffer solutionbefore use.

The hydrogel disks were immersed into dioxane of 2 mg/ml NHS and 2 mg/mlEDC in dioxane/water (14:1) mixture for 1 hour at room temperature. Thehydrogel disks shrank during the soaking with the dioxane/watersolution. The disks were removed from the solution, soaked in Milliporewater to swell them back, rinsed with Millipore water, and soaked in PBSbuffer for another 30 min. The samples were immersed in a 100 μg/mLfibronectin solution at 4° C. for 24 hours.

Bovine aortic endothelial cells (BAECs) were maintained in continuousgrowth in Dulbecco's modified Eagle's medium (DMEM), supplemented with10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential aminoacids, and 2% penicillin streptomycin solution at 37° C. in a humidifiedatmosphere containing 5% CO₂ on tissue culture polystyrene flasks. BAECswere removed from the flask surfaces by washing twice with 10 mL of PBSfollowed by incubation in 2 mL of trypsin/ethylenediamine tetraaceticacid (0.05%/0.53 mM) for detachment. After cells were detached, cellswere then resuspended in 8 mL of DMEM, and the suspension wascentrifuged at 1000 rpm for 5 min. The supernatant was removed and thecells were diluted in DMEM with 5% or 10% FBS at a final concentrationof 105 cells/mL. The hydrogel disks were washed by PBS in a 24-wellplate and 2 mL cell suspension was added to each well. The cells werethen incubated with the samples for 3 days at 37° C. in a humidifiedatmosphere of 5% CO₂. The morphology and proliferation of the cells wereobserved using a Nikon TE200 phase contrast microscope equipped with adigital camera using a 10× objective between 2 hours and 3 days ofcultivation. Cell seeding density was determined using a hemocytometer.Cell culture medium and reagents were obtained from Gibco (Gaithersburg,Md.).

Example 5 Representative CBAA-Based Protein Array

Protein microarrays on polyCBAA surfaces were made using a microcontactprinting robot (Spotbot, Telechem Inc.) to monitor specific proteinbinding events using a surface plasmon resonance (SPR) imaging sensorwith polarization contrast. The array is illustrated schematically inFIG. 13A. The SPR imaging sensor is based on intensity modulation, whichallows measurements of spatially resolved real-time changes of SPRsignals. FIG. 14 shows three sensorgrams corresponding to proteinadsorption from three complex matrices (100% sera, 100% plasma, and 1mg/ml fibrinogen), each flowed for five minutes at 30 microliters perminute on the CBAA surface. In the sensorgrams, the first five minutesis a baseline established by flowing phosphate buffered saline (PBS) pH7.4, followed by the addition of each of the complex media. The complexmedia have a different RI causing a significant shift in the SPR. Afterfive minutes the solution is changed back to PBS, so that the backgroundrefractive index is now identical to that during the collection of theoriginal baseline. Any shift in the baseline before and after theaddition of the complex media represents a quantitative measurementproteins bound to the surface. From FIG. 14 there was no measurablebinding from 100% plasma or 1 mg/ml fibrinogen, while the 100% serum hasminimal binding.

The CBAA surface was specifically functionalized with two antibodies ina microarray pattern. FIG. 15 shows the SPR image of the antibodies onthe surface. The difference in the brightness of the background and theprotein spots quantitatively represents the amount of proteinsimmobilized. The protein microarray was produced by first activating theCBAA surface by reacting it with a solution containing 0.1M EDC and 0.05M NHS in 100 mM sodium acetate buffer at pH 3.3 for 30 minutes. Using amicrocontact printing robot equipped with 500 μm diameter stealth pins,the anti-human chorionic gonadotropin (hCG) and anti-Salmonellaantibodies with printed onto the activated polymer surface and allowedto react for 30 minutes. Excess protein was then rinsed from the surfaceand activated background was deactivated by soaking the microarray chipin a solution of 100 mM hydroxylamine in 100 mM sodium carbonate bufferat pH 9.0 for about 18 hours, in order to hydrolyze the active NHSesters back to carboxylic acids returning the surface to itszwitterionic state with inherent non-fouling characteristics. FIG. 16shows the specific detection of 10 μg/ml hCG on the spots with anti-hCGwhile no detection is observed on the reference anti-Salmonella spot orthe background. The CBAA surface not only has a nonfouling background,but also can be specifically functionalized with protein enablingdetection of protein-protein binding events. The surface chemistry haswide range of application, especially in biomedical diagnostics, wheredetections are commonly performed in complex biological media likeblood. Many new label-free methods such as SPR have become popular,however without non-fouling background and specific surface,deconvolution of specific and non-specific binding is difficult.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A crosslinked polymerhaving a plurality of target binding partners covalently coupledthereto, wherein the crosslinked polymer comprises a plurality ofcarboxylic acid groups and a plurality of positive charged groups,wherein the crosslinked polymer is substantially electronically neutral,and wherein the target binding partner has affinity toward a targetmolecule.
 2. The crosslinked polymer of claim 1, wherein the crosslinkedpolymer is a crosslinked poly(carboxybetaine).
 3. The crosslinkedpolymer of claim 2, wherein the crosslinked poly(carboxybetaine) isprepared from one or more monomers selected from the group consisting ofcarboxybetaine acrylates, carboxybetaine acrylamides, carboxybetainevinyl compounds, carboxybetaine epoxides, and mixture thereof.
 4. Thecrosslinked polymer of claim 2, wherein the target binding partner isselected from the group consisting of enzymes, antibodies, antigens,receptors, and ligands.
 5. The crosslinked polymer of claim 2, whereinthe crosslinked polymer is a hydrogel.